P794: DIFFERENTIATION POTENTIAL AND RELATIVE GENE EXPRESSION LEVELS ARE CHANGED IN CFU-F FROM BONE MARROW OF APLASTIC ANEMIA PATIENTS AT THE ONSET OF THE DISEASE
Authors: A. Dorofeeva, I. Shipounova, A. Luchkin, Z. Fidarova, E. Mikhailova
Journal: HemaSphere (2022)

Background: Aplastic anemia (AA) is a heterogeneous blood system disease characterized by the bone marrow (BM) aplasia in and pancytopenia. There are non-severe (NAA), severe (SAA) and very severe (VSAA) forms of AA. The main cause of AA is believed to be the damage to the hematopoietic stem cells (HSC) by autoreactive cytotoxic T-lymphocytes. Another reason for the AA development may be a dysfunction of the BM stromal microenvironment. One of the ways of study the BM stroma is the analysis of colony-forming units of fibroblasts (CFU-F). Aims: To determine the CFU-F concentration in BM of AA patients, to assess the differentiation potential of individual colonies and the expression level of genes associated with proliferation, differentiation, immune response, and maintenance of HSC in CFU-F from BM of untreated AA patients. Methods: The study included 38 AA (15 with SAA, 4 with VSAA and 19 with NAA) patients in the debut. VSAA was combined with SAA for the analysis. The control group included 22 healthy donors. BM was aspirated at diagnostic punctures in patients and planned exfusion from donors after informed consent. CFU-F cultures were obtained according to the standard method. The colonies number was counted, and total RNA was isolated. Individual CFU-F colonies were obtained by plating 15000 BM mononuclear cells per well of a 96-well plate in aMEM with 20% FBS. Wells with individual clones had been analyzed further. The differentiation potential of CFU-F colonies was determined by the level of marker gene expression after incubation in a medium with inducers of osteogenic or adipogenic differentiation. Gene expression was determined by TaqMan RT-PCR. Data are presented as mean±standard error of the mean. Differences were considered significant at p<0.05 using an unpaired Student’s t-test. Results: CFU-F concentration in the BM of AA patients was not altered (33.58±9.12 per 106 BM mononuclear cells versus 34.43±8.39 in donors). The differentiation potential of CFU-F clones from SAA patients was skewed to osteogenesis when compared with donors and NAA patients. The proportion of CFU-F clones that failed to differentiate was decreased in SAA patients, while in NAA patients the proportion of such clones increased in comparison with healthy donors. These data may indicate a change in the ratio of stromal precursors of different maturity in the BM of patients with NAA and SAA compared with healthy donors (Table 1). CFU-F cultures of SAA patients significantly differed from those of healthy donors in the expression level of 10 genes (Table 2). The similar changes in 5 of these genes (PDGFRA, PDGFRB, ANG1, CFH, NES) were also found in NAA, but, with the exception of NES, the changes were not significant. Most of these genes are associated with the HSC maintenance, thus, the identified changes could be the result of BM aplasia. The expression level of the other 5 genes in CFU-F of NAA patients did not differ from that in donors (FGFR1, IL1B, PPARG, HLA-DR) or tended to change oppositely than in SAA (IL10). These genes are functionally associated with immune response and differentiation. Possibly, the activity of these genes prevents the development of more pronounced aplasia in NAA patients. Image:Summary/Conclusion: CFU-F of NAA and SAA patients differ from CFU-F of healthy donors by their differentiation potential and gene expression. Changes in CFU-F of SAA patients are more pronounced, which may be associated with deeper aplasia. This work was supported by the Russian Foundation for Basic Research, grant no. 19-015-00280.